The objective of the proposed research is to establish the molecular mechanism of NMDA receptor activation and gating. More specifically, these studies will evaluate the hypothesis that agonist binding is couple to the channel gate through the membrane-spanning M3 domain. Receptors will be locked in an activated state via covalent modification and characterized at the single-channel level, confirming that M3 is functionally distinct from the gate. Site-directed mutagenesis will then be used to stabilize the ligand-binding domain (LBD) in the agonist-bound conformation, followed by modification rate studies to link structural changes in the LBD to movement of M3. Further experiments will identify the functional role of the LBD interface, recently shown to control activation in AMPA receptors. Sets of charged residues at the NR1/NR2 interface will be swapped between subunits, and the resulting mutants analyzed for changes in gating properties. In addition, proton dose-response studies will indicate whether the dimer interface is involved in pH regulation. This research will address several unanswered questions regarding the activation, gating, and regulation of the NMDA receptor, improving its effectiveness as a therapeutic target and our understanding of neuronal diseases.